This blog is meant to allow Fragment-based Drug Design Practitioners to get together and discuss NON-CONFIDENTIAL issues regarding fragments.

05 May 2014

Biofragments: extracting signal from noise, and the limits of three-dimensionality

What does this protein do? Now that any genome can be
sequenced, this question gets raised quite often. In many cases it is possible
to give a rough answer based on protein sequence: this protein is a serine
protease, that one is a protein tyrosine kinase, but figuring out the specific
substrates can be more of a challenge. In a recent paper in ChemBioChem, Chris Abell and
collaborators at the University of Cambridge and the University of Manchester
attempt to answer this question with fragments.

The bacterium Mycobacterium
tuberculosis (Mtb), which causes
tuberculosis, has 20 cytochrome P450 proteins (CYPs), heme-containing enzymes
that usually oxidize small molecules. Although some are essential for the
pathogen, it is not clear what many of them do. The researchers used an
approach called “biofragments” to try to pin down the substrate of CYP126.

The biofragments approach starts by selecting a collection of
fragments based on known substrates. Of course, the specific substrates
are not known, so in this case the
researchers started with a set of several dozen natural (ie, non-synthetic)
substrates of various other CYPs, both bacterial and eukaryotic. They then
computationally screened the ZINC database of commercial molecules for
fragments most similar to these substrates and purchased 63 of them. Perhaps
not surprisingly given their similarity to natural products, these turned out
to be more “three-dimensional” than conventional fragment libraries, as
assessed both by the fraction of sp3 hybridized carbons and by
principal moment-of-inertia.

Next, the researchers screened their fragments against
CYP126 using three different NMR techniques (CPMG, STD, and WaterLOGSY). Since
they were primarily interested in hits that bind at the active site, they also
used a displacement assay in which the synthetic heme-binding drug ketoconazole
was competed against fragments. This exercise yielded 9 hits – a relatively
high 14% hit rate.

Strikingly, all of the hits are aromatic, and 7 of them
could reasonably be described as planar. In other words, even though the
biofragment library was relatively 3-dimensional, the confirmed hits were some
of the flattest in the library! The researchers interpreted this to mean that
“CYP126 might preferentially recognize aromatic moieties within its catalytic
site,” but there could be something more general going on – perhaps aromatics
are simply less complex, and thus more promiscuous.

Examining the fragment hits more closely, the researchers
found that one of them – a dichlorophenol – produced a
spectrophotometric shift similar to that produced by substrates when bound to the enzyme. This led them
to look for similar structures among proposed Mtb metabolites. Weirdly, pentachlorophenol came up as a possible
hit, and a spectrophotometric shift assay reveals that this molecule does have
relatively high affinity for CYP126. Whether this is a biologically relevant
substrate for the enzyme remains to be seen.

This is an intriguing approach, but I do have reservations.
First, in constructing fragment libraries based on natural products, it is
essential to avoid anything too “funky”. The Abell lab is one of the top
fragment groups out there, well aware of potential artifacts, and has a long
history of studying CYPs, but researchers with less experience could easily populate a library with dubious compounds.

More fundamentally though, I wonder about the basic premise
of biofragments. The whole point of fragments is that they have low molecular
complexity and are thus likely to bind to many targets, so is it realistic to
try to extract selectivity data from them? Indeed, as we’ve seen (here and
here), fragment selectivity is not necessarily predictive of larger molecules.

That said, the approach is worth trying. Even if it doesn’t
ultimately lead to new insights into proteins’ natural substrates, it could
lead to new inhibitors.

3 comments:

I saw this paper and was intrigued. It is a very similar approach to the Emerald Fragments of Life. This is also something I have done in that past. It is my experience /impression that this is not a fantastic source of fragment hits. I think you would be just as successful starting with a library of all 20 amino acids as with "substrate fragments". Their approach might be useful for a target validation or "What the heck does this enzyme do study", but I am wholly unconvinced that this is a good entree into any sort of fragment diversity.

Although not strictly fragment-based, a colleague in our lab has had success with a related approach. She relied on molecular docking of the entire KEGG library of metabolites and the genome context of the unknown bacterial enzymes to assign function.

http://www.ncbi.nlm.nih.gov/pubmed/24056934

To me, this seems like a kind of in-silico parallel to the experimental approach discussed above. The experimental approach of looking for fragment binders is perhaps handicapped by the small size and diversity of the collection. Docking used in this kind of exploratory analysis is not limited by library size or complexity.